Ku Complex Suppresses Recombination in the Absence of MRX Activity During Budding Yeast Meiosis

BMB Rep. 2019; 52(10): 607-612 BMB www.bmbreports.org Reports

Ku complex suppresses recombination in the absence of MRX activity during budding yeast meiosis

Hyeseon Yun & Keunpil Kim* Department of Life Science, Chung-Ang University, Seoul 06974, Korea

During meiosis, programmed double-strand breaks (DSBs) are DNA repair programs such as homologous recombination and repaired via recombination pathways that are required for non-homologous end joining (NHEJ) that are highly conserved faithful chromosomal segregation and genetic diversity. In in all eukaryotic organisms. The choice of repair program meiotic progression, the non-homologous end joining (NHEJ) between NHEJ and recombination depends on cell cycle pathway is suppressed and instead meiotic recombination phase and the process of DSB ends (5-7). For homologous initiated by nucleolytic resection of DSB ends is the major recombination to proceed to repair spontaneous DNA damage pathway employed. This requires diverse recombinase proteins and meiotic Spo11-catalyzed DSBs, many recombinase and regulatory factors involved in the formation of crossovers proteins and chromosome structural proteins play an (COs) and non-crossovers (NCOs). In mitosis, spontaneous important role. Homologous recombination utilizes the sister DSBs occurring at the G1 phase are predominantly repaired chromatid (or homologs in diploids) as a template for the via NHEJ, mediating the joining of DNA ends. The Ku repair of accidental DSBs during mitosis (8-11). Unlike mitosis, complex binds to these DSB ends, inhibiting additional DSB however, meiotic DSBs are repaired via the interhomolog resection and mediating end joining with Dnl4, Lif1, and recombination pathway to achieve genetic diversity for the Nej1, which join the Ku complex and DSB ends. Here, we next generation. In meiotic recombination, DSB ends report the role of the Ku complex in DSB repair using a predominantly utilize the homologous chromosome as a physical analysis of recombination in Saccharomyces template for strand exchange to produce non-identical gametes cerevisiae during meiosis. We found that the Ku complex is by exchanging genetic information (12). not essential for meiotic progression, DSB formation, joint Meiotic recombination is initiated by programmed DSBs molecule formation, or CO/NCO formation during normal induced by the meiosis-specific topoisomerase II-like protein meiosis. Surprisingly, in the absence of the Ku complex and Spo11 (13). For recombination to progress, the highly functional Mre11-Rad50-Xrs2 (MRX) complex, a large portion conserved Mre11-Rad50-Xrs2 (MRX in yeast; Mre11-Rad50-Nbs2 of meiotic DSBs was repaired via the recombination pathway in mammals) complex binds to DSB regions and controls end to form COs and NCOs. Our data suggested that Ku complex resection to remove Spo11 (14-16). DSB end resection during prevents meiotic recombination in the elimination of MRX S and G2 phases of the cell cycle is achieved through activity. [BMB Reports 2019; 52(10): 607-612] cyclin-dependent kinase (CDK)-mediated phosphorylation of Sae2, an essential factor for activating the DNA endonuclease of the MRX complex, which is associated with bridging DNA INTRODUCTION ends (17-19). Exonucleases, such as Exo1 and Dna2-Sgs1, resect the 5ʹ-ends of DNA strands to generate 3ʹ single-strand DNA double-strand breaks (DSBs) are the most toxic form of DNA (ssDNA) that is required for recombinase binding and DNA lesions generated by various types of DNA damaging homology searching (20). Replication protein A (RPA)—a agents, such as free radicals, ultraviolet light, and ionizing heterotrimeric complex consisting of Rfa1, Rfa2, and radiation (1-4). To repair DSBs, cells induce tightly regulated Rfa3—binds to the ssDNA of DSB ends to inhibit secondary structures formed by ssDNA self-complementizing or to *Corresponding author. Tel: +82-2-820-5792; Fax: +82-2-820-5206; protect DSB ends from degradation (21). After displacement of E-mail: [email protected] RPA from ssDNA, Rad51, a RecA homolog, forms nucleofilaments that are also used for homology searching and https://doi.org/10.5483/BMBRep.2019.52.10.245 homolog pairing during mitosis. However, in meiotic recombination, Rad51 functions as an auxiliary factor of Dmc1 Received 21 October 2018, Revised 6 December 2018, for homolog bias (9). Accepted 25 January 2019 NHEJ, a prominent DSB repair pathway of the mitotic cell Keywords: Double-strand breaks, Homologous recombination, Ku cycle, mediates direct re-ligation of DSB ends from complex, Meiosis, Non-homologous end-joining spontaneous DNA damage. NHEJ is initiated by a DNA end

ISSN: 1976-670X (electronic edition) Copyright ⓒ 2019 by the The Korean Society for Biochemistry and Molecular Biology This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/li- censes/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Activation of the Ku complex in meiosis Hyeseon Yun and Keunpil Kim

binding complex, the Ku70-Ku80 heterodimeric complex (Ku complex), which prevents 5’ strand resections of DSB ends (17, 22). Once the Ku complex binds to the DSB ends, it serves as a core site of NHEJ accessory factor recruitment to the DNA breaks. Inaccurate end-joining as a result of Ku complex-deficiency causes chromosomal breaks and aneuploidy (23). In budding yeast, DNA end processing involved in the NHEJ pathway is mediated by diverse factors including Dnl4 (ATP-dependent ligase; DNA ligase IV in vertebrates), Lif1 (XRCC4 in vertebrates), Nej1 (XLF in vertebrates), and Pol4 (Pol  and Pol  in vertebrates) (24-26). Haber and colleagues reported that diploid yeast cells suppressed expression of Nej1 and Lif1, but ectopic expression of Nej1 restored NHEJ pathway to diploid cells (27). The DNA end bridge complex is targeted by a DNA ligase complex that mediates end-joining of Fig. 1. Ku70 is not essential for meiotic progression. (A) Meiotic DSB ends and inhibits DSB end resection, which is processed progression of WT and ku70 cells. Meiosis was induced in by nuclease-helicase enzymes (24-26). Finally, Pol4 and Lig4 synchronized yeast in SPM and cell divisions were counted at the indicated time points. The error bar represents the standard are required for filling in the DNA gaps (25). In mammalian deviation (SD; n = 3). (B) Representative images of DAPI-stained cells, DSB repair via homologous recombination utilizes the nuclei of WT and ku70 strains cultured in SPM for 24 h. Scale sister chromatid as a template because it is nearby during the bar = 2.5 m. (C) Analysis of spore viability in WT and ku70 ＞ S/G2 phase, while NHEJ is the major DSB repair process that strains (n 100). (D) MMS sensitivity test. Cells were serially diluted and spotted onto YPD plates and YPD plates containing occurs in all cell cycle phases. The Ku complex binds to a 0.01% and 0.03% MMS. DNA end to form the Ku:DNA complex that serves as a platform where a ligase complex including XLF, XRCC4, and DNA ligase IV can dock to rejoin the ends (25, 26). three spores, respectively, compared with the 92% and 3% of Here, we investigated the role of the Ku complex and the WT, respectively (Fig. 1B and 1C). Thus, ku70 cells under- relationship between the Ku and MRX complexes. Ex- went meiosis normally and formed viable spores as did the perimental studies of NHEJ-mediated meiotic DSB repair are WT, confirming that NHEJ is not an essential pathway for challenging because recombination is the major DSB repair repairing Spo11-induced DSBs. To understand the role of the pathway in meiosis. To this end, we present a NHEJ- Ku complex in mitotic DNA damage repair, we employed the independent role of the Ku complex in meiotic recombination methyl methane sulfonate (MMS) sensitivity test in the absence of Saccharomyces cerevisiae through physical analysis of of the Ku complex (Fig. 1C and 1D). ku70 cells grew at recombination. similar levels as the WT in YPD media containing 0.01% and 0.03% MMS (Fig. 1D). Thus, DNA damage of vegetative cells RESULTS is not lethal for ku70 mutants, indicating that NHEJ is not an essential pathway in MMS-induced DSB repair. The Ku complex is not essential for division and spore viability in normal meiosis Physical analysis of meiotic recombination The Ku complex rapidly localizes to DSB sites and is involved To determine the molecular pathway involved in meiotic in protecting DNA ends from nuclease-helicase processing as recombination, we monitored recombination intermediates well as recruiting NHEJ proteins (25). To provide insights into and final outcomes (crossovers [COs] and non-crossovers the role of the Ku complex during meiosis, we observed [NCOs]) using the HIS4LEU2 assay system for chromosome III meiotic division and spore formation in wild-type (WT) and (Fig. 2). In the HIS4LEU2 assay system, COs and NCOs can be ku70 mutant cells (Fig. 1A and 1B). Meiosis was induced in detected after digesting genomic DNA with XhoI and NgoMIV cells incubated in sporulation medium (SPM) that were then enzymes. After synchronizing yeast cells at the G1 phase in harvested from the culture at different time points (0, 2.5, 3.5, pre-sporulation medium (SPS), the cells were transferred to 4, 5, 6, 7, 8, 10, and 24 h). In WT cells, meiotic division sporulation medium to initiate meiosis. Cells were then treated began after 5 h in sporulation media and rapidly progressed with psoralen after harvesting to produce interstrand-crosslink with 50% of cells having underwent division after DNA, which stabilizes single-end invasions (SEIs) and approximately 6 h. In ku70 cells, normal nuclear division double-Holliday junctions (dHJs; 8-10, 28, 29). Meiotic DNA occurred with a slight delay of about 20 min compared with samples were digested with XhoI and then DNA fragments that of WT (Fig. 1A). Moreover, DAPI staining indicated that were analyzed by DNA gel electrophoresis and Southern both WT and ku70 cells exhibited normal nuclei separation blotting using Probe A (Fig. 2A and 2B). DNA species of after 24 h; 91.4% and 4.2% of ku70 cells produced four and interest, DSBs and COs, were quantified using a phospho-

608 BMB Reports http://bmbreports.org Activation of the Ku complex in meiosis Hyeseon Yun and Keunpil Kim

Fig. 3. The absence of Ku70 reduces DSB levels in a rad50S background. (A) MMS sensitivity test of rad50S and rad50S ku70 cells. (B) MMS sensitivity test of rad50and rad50ku70 cells. (C) 1D gel electrophoresis of rad50S and rad50S ku70 of the HIS4LEU2, ARG4, BUD23, and CYS3 loci. (D) Maximum level of DSBs at each hotspot locus. Error bars represent SD (n = 3).

experiments are presented in Supplemental Fig. 1. The Fig. 2. Normal progression of meiotic DSB repair and formation of COs and NCOs in the absence of Ku70. (A) Physical map of maximum level of COs and NCOs in WT cells was 3.8% and the recombination assay for chromosome III. The HIS4LEU2 3.1%, respectively. The levels and turnover of COs and NCOs hotspot schematic includes restriction enzyme polymorphisms and in ku70 cells were similar to those of WT cells, consistent the Southern blot probe (probe A). (B) 1D gel electrophoresis of with our meiotic division findings (Fig. 1 and Fig. 2G). To WT and ku70 strains. Cells were harvested at different time points (0, 2.5, 3.5, 4, 5, 6, 7, 8, 10, and 24 h). (C) Quantitative examine whether the Ku complex affects homolog bias, we analysis of DSBs in WT and ku70 cells. (D) Structure of the 2D performed 2D gel electrophoresis (Fig. 2E). Several types of gel analysis of the HIS4LEU2 locus. (E) 2D gel electrophoresis of JMs were detected using 2D gel analysis including intersister WT cells. The average ratio of IH:IS-dHJ was 5:1 for both WT SEIs (IS-SEIs), IH-SEIs, IH-dHJs, and IS-dHJs. Consistent with and ku70 cells. (F) Gel analysis of COs and NCOs. (G) Quantification of IH-COs and IH-NCOs in WT and ku70 cells. our previous results for WT cells, IH-dHJ levels were higher Error bars represent SD (n = 3) and significance was determined than IS-dHJs at a ratio of 5:1 (Fig. 2E). Moreover, the ratio of by a Student t test. Maternal species, paternal species, DSBs, IH-dHJs and IS-dHJs in ku70 cells was also 5:1. Additionally, double-strand breaks; IH-COs, interhomolog crossovers; IH-NCOs, IH-SEIs occurred at high levels in both WT and ku70 cells. interhomolog non-crossovers. SEI, single end invasion; IH-dHJ, interhomolog-double Holliday junction; IS-dHJ, intersister-double Thus, the results indicate that the Ku complex is not required Holliday junction. for the formation of JMs (DSB-to-JM transition) and establishment of homolog bias. image analyzer. Parental DNA species were detected at 5.9 kb DSBs levels are reduced at the HIS4LEU2, ARG4, BUD23, for maternal chromosomes and 4.3 kb for paternal and CYS3 loci in rad50S ku70 cells chromosomes (Fig. 2A and 2B). DSB signals appeared at 3.0 In rad50S mutant cells, the MRX complex is inactivated and kb and 3.3 kb in one-dimensional (1D) gel electrophoresis. thus DSBs accumulate instead of forming CO and NCO Native/native two-dimensional (2D) gel electrophoresis was recombinants (30). Thus, the total number of DSBs can be performed to detect joint molecules (JMs; SEI and dHJ; Fig. 2D measured from the rad50S allele, which is blocked at the and 2E). COs and NCOs were distinguished in 1D gels at 4.6 DSB-to-JM transition. Surprisingly, the rad50S cells exhibited kb and 4.3 kb, respectively (Fig. 2A and 2F). strong MMS sensitivity, but the ku70 mutation partially suppressed DNA damage of rad50S cells (Fig. 3A). Similar Repair of meiotic double-strand breaks progressed normally results were obtained when rad50 and rad50 ku70 cells in ku70 cells were examined in the same experiments (Fig. 3B). These In WT cells, DSBs were initiated after 2.5 h and peaked at 4 h results indicate that damaged DNA is possibly repaired in the with approximately 16.7% hybridizing DNA species that then deficiency of Ku complex and DSB resection. To investigate disappeared after 6 h. DSB levels and turnover were similar whether the Ku complex is required for DSB formation, we between WT and ku70 cells (Fig. 2B and 2C). Similar data for used 1D gel electrophoresis for rad50S DSB analysis at the another set of physical analysis of independent time course HIS4LEU2, ARG4, BUD23, and CYS3 loci (Fig 3C and 3D). http://bmbreports.org BMB Reports 609 Activation of the Ku complex in meiosis Hyeseon Yun and Keunpil Kim

Notably, total levels of DSBs in rad50S ku70 cells were DSBs progressed to COs at a much later time point (Fig. 4A). lower than those of WT cells at all loci. Furthermore, a This finding suggests that the COs detected in rad50S ku70 significant subset of DSBs in rad50S ku70 cells were repaired cells may result from meiotic recombination, implying that to form COs at the HIS4LEU2 hotspot, which can distinguish nucleolytic resection of DSB ends occurred in the absence of between IH-COs and IH-NCOs (Fig. 4A and 4B). Thus, a the Ku complex and a functional MRX complex. We further portion of DSBs in rad50S ku70 cells progressed to form COs investigated the formation of COs and NCOs in rad50S ku70 at a later time point, indicating that cells repaired DSBs via the mutant cells. Notably, COs and NCOs were detected in recombination pathway. rad50S ku70 cells (Fig. 4B and 4C). In rad50S ku70, NCOs appeared after approximately 8 h and COs appeared after 10 Ku70 is involved in DSB repair during arrest of the DSB end h, indicating that NCOs formed earlier than COs. Interestingly, resection process the maximum levels of COs were attained by 24 h. Therefore, At the HIS4LEU2 locus in rad50S ku70 cells, a subset of our findings indicate that DSB repair occurred to form CO and NCO through meiotic recombination in rad50S ku70 mutant cells.

DISCUSSION

DSBs can arise from diverse reactive metabolites, ionizing radiation, or stalling of DNA replication during cell cycle. Inappropriate repair of DSBs leads to cell death, senescence, or cancer. Two distinct DNA repair pathways, NHEJ and homologous recombination, eliminate DSBs depending on the cell cycle phase or the nature of DSB end process. During meiosis, cells induce programmed DSBs that are generated by Spo11 and accessory factors. The post-DSB role of Exo1 and the MRX complex is essential for promoting recombination. It has been reported that the MRX complex, in coordination with Sae2, mediates ssDNA nick formation and exhibits 3’ to 5’ exonuclease activity that resects the ssDNA towards Spo11-binding regions. Additionally, Exo1 and the Dna2-Sgs1 complex promote formation of long stretches of ssDNA that can be used for homology searching on homologous chromosomes during meiosis. The long single-stranded overhangs of DSBs are bound by the homology search and strand exchange proteins Rad51, Dmc1, and accessory factors including Rad52, Rad54, Rad54, Rad57, the PCSS complex, Hed1, Rdh54/Tid1, Hop-Mnd1, and Mei5-Sae3 (21). The MRX/N complex has been implicated in NHEJ-mediated DSB repair during mitosis in budding yeast. However, Ku complex- mediated NHEJ is dispensable in meiotic recombination of budding yeast (Fig. 4D), whereas it is essential for the Fig. 4. Absence of Ku70 and a functional MRX complex successful maintenance of genomic integrity in mammalian promotes CO and NCO formation. (A) Representative images of cells (26). The absence of NHEJ and a functional MRX CO and NCO gels in rad50S and rad50S ku70. (B) Quantitative complex in Caenorhabditis elegans channeled meiotic DSB analysis of COs and NCOs in rad50S and rad50S ku70 cells. Error bars represent SD (n = 3). (C) Maximum level of COs and repair to the exonuclease-dependent recombination pathway NCOs in rad50S and rad50S ku70 cells. Error bars represent SD from NHEJ pathway (31). The absence of an MRX complex (n = 3) and significance was determined by a Student t test. **P showed no meiotic DSB-to-JM transition or CO and NCO ＜＜ 0.01, ***P 0.001. (D) Proposed model for the roles of the recombinants, as evidenced by physical analysis of recom- MRX complex and NHEJ pathway in meiotic recombination. DSBs are catalyzed by Spo11 and the MRX complex plays a role in bination in budding yeast. The presence of unprocessed DSBs DNA resection and Spo11-oligonucleotide release (34). Exo1 and induces a checkpoint signal requiring pachytene checkpoint the Dna2-STR complex promote additional DSB end resection to protein 2 (Pch2) that functions with Tel1 and the MRX create long ssDNA overhangs. In the absence of NHEJ and a complex (32). Thus, we theorized that the MRX complex functional MRX complex, ∼50% of DSBs progressed to recombination to form COs and NCOs (this study). STR, possibly acts together with Pch2 to promote normal meiotic Sgs1-Top3-Rmi1; SDSA, synthesis-dependent strand annealing. recombination. Herein, we observed diverse recombination

610 BMB Reports http://bmbreports.org Activation of the Ku complex in meiosis Hyeseon Yun and Keunpil Kim

phenotypes as follows, (i) meiotic recombination and nuclear then stained with DAPI (1 l/ml) and the nuclei were counted division progressed normally in the absence of Ku70 as in WT (n = 200). Nuclei stained with DAPI were observed using cells; (ii) DSB levels were found reduced at various loci of fluorescence microscopy (Eclipse Ti-E; Nikon, Tokyo, Japan) yeast chromosomes in rad50S ku70 cells; (iii) a large portion and imaged using the Nikon DS-Qi2. of DSBs formed CO and NCO recombinants in rad50S Ku70 during meiosis; and (iv) a subset of DSBs remained unrepaired Meiotic time course analysis for 24 h. It has been also reported that NHEJ is suppressed by Meiotic time course was performed as described previously repression of Nej1 in diploid yeast cells (27). Our results (8-11). Cells were streaked onto YPG plates (1% bacto yeast further indicate that Ku complex is not required for extract, 2% bacto peptone, 2% bacto agar, and 3% glycerol) recombination during normal meiosis. Moreover, in the and incubated overnight. Cells were diluted onto YPD plates absence of a functional NHEJ and MRX complex, and incubated for 2 days. Single colonies were then incubated recombination occurred, leading to CO and NCO formation. in YPD liquid media for 18 h. To synchronize cells in G1 In WT cells, the Ku complex was not essential for CO and phase, a 1/500 dilution of YPD culture was added to SPS NCO formation, as the MRX complex and Exo1/Dna2-Sgs1 (0.5% bacto yeast extract, 1% bacto peptone, 1% potassium function in forming ssDNA overhangs of DSBs (33, 34). When acetate, 0.05 M potassium biphthalate, 0.5% ammonium both the MRX complex and Ku complex were defective, sulfate, and 0.17% yeast nitrogen base without amino acids; Exo1/Dna2-Sgs1 may have functioned to expose ssDNA pH 5.5) in a shaking incubator for 18 h. Synchronized cells through their 5’ end resection activity, although this activity were then transferred to SPM. Meiotic cells were harvested at was not fully active without the initial strand nicking by the different time points and crosslinked with psoralen MRX complex (Fig. 4D). (Sigma-Aldrich, St. Louis, MO) using ultraviolet light at 360 In the present study, we found that the Ku complex is nm for 15 mins. involved in meiotic DSB repair in the absence of MRX activity, but not the presence of the MRX complex. These findings are Physical analysis of meiotic recombination important for understanding how cells deal with programmed Physical analysis was performed as described previously (8, 9). DSBs (or endogenous damage-induced DSBs) during meiosis Detailed information regarding the procedures is described in and how defective DSB end resection affects meiotic Supplemental Materials. recombination in the presence or absence of another repair pathway (35). ACKNOWLEDGEMENTS

MATERIALS AND METHODS This work was supported by grants to K.P.K. from the National Research Foundation of Korea (NRF) funded by the Korean Yeast strains Ministry of Science, ICT and Future Planning (MSIT; We used the Saccharomyces cerevisiae SK1 strain in this NRF-2017R1A2B2005603; NRF-2018R1A5A1025077) and the study. Detailed information regarding strains is listed in Next-Generation BioGreen 21 Program (SSAC; No. PJ01322801), Supplemental Materials Table S1. Rural Development Administration, Republic of Korea.

MMS sensitivity test CONFLICTS OF INTEREST Cell were grown in YPD liquid medium (1% bacto yeast extract, 2% bacto peptone, and 2% glucose) for 24 h. Cells The authors have no conflicting interests. were diluted 10−1, 10−2, 10−3, 10−4, and 10−5 in distilled water and spotted on YPD plates (1% bacto yeast extract, 2% REFERENCES bacto peptone, 2% bacto agar, and 2% glucose) and YPD plates containing 0.01% and 0.03% MMS. The plates were 1. Chung WH (2014) To peep into Pif1 helicase: then incubated for 2 days. Multifaceted all the way from genome stability to repair-associated DNA synthesis. J Microbiol 52, 89-98 Spore viability test 2. Choi DH, Lee R, Kwon SH and Bae SH (2013) Hrq1 Diploid cells were grown in SPM (1% potassium acetate, functions independently of Sgs1 to preserve genome 0.02% raffinose, and 0.01% antifoam) for 24 h. Spores were integrity in Saccharomyces cerevisiae. J Microbiol 51, plated onto YPD plates through tetrad dissection and then 105-112 3. Sung P (2018) Introduction to the Thematic Minireview incubated for 2 days. Series: DNA double-strand break repair and pathway choice. J Biol Chem 293, 10500-10501 Meiotic division 4. Haber JE (2018) DNA Repair: The Search for Homology. Cells in SPM were harvested at different time points, fixed in BioEssays 40, 1700229 sorbitol solution (40% ethanol and 0.1 M sorbitol). Cells were 5. Fouquin A, Guirouilh-Barbat J, Lopez B, Hall J, http://bmbreports.org BMB Reports 611 Activation of the Ku complex in meiosis Hyeseon Yun and Keunpil Kim

Amor-Guéret M and Pennaneach V (2017) PARP2 The two ends of a double strand break. Mutat Res 809, controls double-strand break repair pathway choice by 70-80 limiting 53BP1 accumulation at DNA damage sites and 22. Tsukamoto Y, Kato J and Ikeda H (1996) Hdf1, a yeast promoting end-resection. Nucleic Acids Res 45, 12325- Ku-protein homologue, is involved in illegitimate 12339 recombination, but not in homologous recombination. 6. Daley JM, Laan RLV, Suresh A and Wilson TE (2005) Nucleic Acids Res 24, 2067-2072 DNA Joint dependence of Pol X family polymerase action 23. Gu Y, Jin S, Gao Y, Weaver DT and Alt FW (1997) in nonhomologous end joining. J Biol Chem 280, Ku70-deficient embryonic stem cells have increased 29030-29037 ionizing radiosensitivity, defective DNA end-binding 7. Cannavo E, Johnson D, Andres SN et al (2018) Regulatory activity, and inability to support V(D)J recombination. Proc control of DNA end resection by Sae2 phosphorylation. Natl Acad Sci U S A 94, 8076-8081 Nat Commun 9, 4016 24. Sorenson KS, Mahaney BL, Lees-Miller SP and Cobb JA 8. Kim KP, Weiner BM, Zhang L, Jordan A, Dekker J and (2017) The non-homologous end-joining factor Nej1 Kleckner N (2010) Sister cohesion and structural axis inhibits resection mediated by Dna2–Sgs1 nuclease- components mediate homolog bias of meiotic recom- helicase at DNA double strand breaks. J Biol Chem 292, bination. Cell 143, 924-937 14576-14586 9. Hong S, Sung Y, Yu M, Lee M, Kleckner N and Kim KP 25. Pannunzio NR, Watanabe G and Lieber MR (2018) (2013) The logic and mechanism of homologous Nonhomologous DNA end-joining for repair of DNA recombination partner choice. Mol Cell 51, 440-453 double-strand breaks. J Biol Chem 293, 10512-10523 10. Hong S and Kim KP (2013) Shu1 promotes homolog bias 26. Lieber MR (2010) The mechanism of double-strand DNA of meiotic recombination in Saccharomyces cerevisiae. break repair by the nonhomologous DNA end-joining Mol Cells 36, 446-454 pathway. Ann Rev Biochem 79, 181-211 11. Yoon SW, Lee MS, Xaver M et al (2016) Meiotic prophase 27. Valencia M, Bentele M, Vaze MB et al (2001) NEJ1 roles of Rec8 in crossover recombination and chromosome controls non-homologous end joining in Saccharomyces structure. Nucleic Acids Res 44, 9296-9314 cerevisiae. Nature 414, 666-669 12. Marcon E and Moens PB (2005) The evolution of meiosis: 28. Lee MS, Yoon SW and Kim KP (2015) Mitotic cohesin Recruitment and modification of somatic DNA-repair subunit Mcd1 regulates the progression of meiotic proteins. Bioessays 27, 795-808 recombination in budding yeast. J Microbiol Biotechnol 13. Lam I and Keeney S (2015) Mechanism and regulation of 25, 598-605 meiotic recombination initiation. Cold Spring Harb 29. Cho HR, Kong YJ, Hong SG and Kim KP (2016) Hop2 and Perspect Biol 7, a016634-a016634 Sae3 are required for Dmc1-mediated double-strand break 14. Gobbini E, Cassani C, Villa M, Bonetti D and Longhese repair via homolog bias during meiosis. Mol Cells 39, MP (2016) Functions and regulation of the MRX complex 550-556 at DNA double-strand breaks. Microbial Cell 3, 329-337 30. Hong S, Choi EH and Kim KP (2015) Ycs4 is required for 15. Seeber A, Hegnauer AM, Hustedt N et al (2016) RPA efficient double-strand break formation and homologous mediates recruitment of MRX to forks and double-strand recombination during meiosis. J Microbiol Biotechnol 25, breaks to hold sister chromatids together. Molecular Cell 1026-1035 64, 951-966 31. Yin Y and Smolikove S (2013) Impaired resection of 16. Chen H, Donnianni RA, Handa N et al (2015) Sae2 meiotic duble-strand breaks channels repair to non- promotes DNA damage resistance by removing the homologous end joining in caenorhabditis elegans. Mol Mre11–Rad50–Xrs2 complex from DNA and attenuating Cell Biol 33, 2732-2747 Rad53 signaling. Proc Natl Acad Sci U S A 112, E1880- 32. Ho HC and Burgess SM (2011) Pch2 Acts through Xrs2 E1887 and Tel1/ATM to modulate interhomolog bias and 17. Hefferin ML and Tomkinson AE (2005) Mechanism of checkpoint function during meiosis. PLoS Genet 7, DNA double-strand break repair by non-homologous end e1002351 joining. DNA Repair 4, 639-648 33. Hunter N (2006) Meiotic Recombination. molecular 18. Huertas P and Jackson SP (2009) Human CtIP mediates genetics of recombination, Topics in Current Genetics, cell cycle control of DNA end resection and double Springer-Verlag, Heidelberg, 381-442 strand break repair. J Biol Chem 284, 9558-9565 34. Neale M, Pan J and Keeney S (2005) Endonucleolytic 19. Huertas P, Cortés-Ledesma F, Sartori AA, Aguilera A and processing of covalent protein-linked DNA double-strand Jackson SP (2008) CDK targets Sae2 to control DNA-end breaks. Nature 436, 1053-1057 resection and homologous recombination. Nature 455, 35. Devkota S (2018) The road less traveled: strategies to 689-692 enhance the frequency of homology-directed repair (HDR) 20. Mimitou EP and Symington LS (2011) DNA end resection for increased efficiency of CRISPR/Cas-mediated trans- - unraveling the tail. DNA Repair (Amst) 10, 344-348 genesis. BMB Rep 51, 437-443 21. Kim KP and Mirkin EV (2018) So similar yet so different:

612 BMB Reports http://bmbreports.org